We investigate the molecular mechanisms by which transmembrane proteins are sorted to different compartments of the endomembrane system such as endosomes, lysosomes and a group of cell-type-specific organelles known as lysosome-related organelles (e.g., melanosomes and platelet dense bodies). Sorting is mediated by recognition of signals present in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats. Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA1, GGA2 and GGA3 proteins (GGAs), and the heteropentameric retromer complex. Proper sorting requires the function of additional components of the trafficking machinery that mediate vesicle tethering and fusion, such as the heterotetrameric GARP complex. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, and investigating human diseases that result from genetic defects (Hermansky-Pudlak syndrome;Alzheimer's disease;developmental disorders) or pathogens'(HIV-1) exploitation of these proteins. Genetic model systems have allowed us to determine the functions of AP complexes in organismal physiology and development. Studies in Drosophila revealed that the GGAs and AP-1 are required for receptor-mediated sorting of acid hydrolases to lysosomes. Other studies performed in collaboration with Eric Everett (University of North Carolina) showed that disurption of the gene encoding the beta2 subunit of AP-2 results in non-syndromic cleft palate. This phenotype is in contrast to that resulting from ablation of the gene encoding the mu2 subunit of AP-2, which is embryonic lethal. The explanation for the different outcomes of these two mutations is that the beta1 subunit of AP-1 can substitute for beta2, whereas the mu1 subunit of AP-1 cannot substitute for the mu2 subunit of AP-2. The absence of beta2 could impair TGF-beta signaling during development of the palate. These observations shed light on the pathogenesis of this common birth defect. The AP-4 complex is the most-recently discovered and least understood of the family of AP complexes. In recent work, we have found that AP-4 interacts with the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP). Disruption of the AP-4-APP interaction shifts the distribution of APP from endosomes to the trans-Golgi network (TGN) and enhances gamma-secretase-catalyzed processing of APP to the pathogenic amyloid-beta peptide. These findings establish AP-4 as a novel regulator of APP processing and trafficking, and as a potential risk factor for Alzheimer's disease. The sorting of newly-synthesized acid hydrolases to lysosomes relies on mannose 6-phosphate receptors (MPRs), which bind the hydrolases at the TGN and transport them to endosomes. The hydrolases are subsequently delivered to lysosomes whereas the receptors return to the TGN for additional rounds of sorting. In previous work, we showed that selection of MPRs for retrieval to the TGN is mediated by the retromer complex. We have recently identified another complex named GARP, which enables tethering and fusion of MPR-containing carriers to the TGN. We had previously shown that the human GARP complex comprises three subunits named VPS52, VPS53 and VPS54. We have now discovered that a protein previously named ANG2/Fat-free is a fourth subunit of the GARP complex. Interference with any of the GARP subunits blocks the delivery of cargos such as MPRs and Shiga toxin from endosomes to the TGN. As a consequence, acid hydrolases are missorted to the extracellular space, lysosomes become dysfunctional, and cells exhibit defective lipid traffic and autophagy. We have also elucidated the structural basis for motor neuron degeneration in the VPS54-mutant wobbler mouse, an animal model for amyotrophic lateral sclerosis (ALS). In collaboration with Aitor Hierro (CIC-bioGUNE, Bilbao, Spain), we solved the crystal structure of the C-terminal domain of VPS54, which harbors the wobbler mutation (leucine-967 to glutamine). The structure revealed that GARP is related to other multisubunit tethering complexes such as the exocyst and COG. In addition, we observed that the wobbler mutation destabilizes VPS54, resulting in its enhanced degradation and decreased levels in all tissues of the mouse. Motor neuron degeneration in this mouse therefore results from decreased levels of GARP. Finally, in another project, we definitively established that the BLOC-3 complex that is defective in some forms of the pigmentation and bleeding disorder, Hermansky-Pudlak syndrome, consists of a 1:1 assembly of two subunits, HPS1 and HPS4. In addition, we demonstrated that this complex interacts with the GTP-bound (active) form of the Rab9 GTPase, suggesting that it might function as a Rab9 effector in the biogenesis of melanosomes and platelet dense bodies.